Integrins are a super-family of cell surface adhesion receptors which control the attachment of cells with the solid extracellular environment both to the extracellular matrix (ECM), and to other cells. Adhesion is of fundamental importance to a cell; it provides anchorage and cues for migration and signals for growth and differentiation. Integrins are directly involved in numerous normal and pathological events, such as cell attachment, migration to blood clotting, inflammation, embryogenesis or cancer growth and metastasis, and as such, they are primary targets for therapeutic intervention.
Integrins are heterodimeric integral transmembrane glycoproteins, consisting of non-covalently linked α and β subunits. The integrins are classified in four overlapping subfamilies, containing the β1, β2, β3 or αV chains, and a particular cell may express several different integrins from each subfamily.
The last decade has shown that integrins are major receptors involved in cell adhesion and so may be a suitable target for therapeutic intervention. Reports concerning integrins are given, e.g., by E. Ruoslahti (J. Clin. Invest., 1991, 87) and R. O. Hynes (Cell, 1992, 69).
The αV-series integrins are now seen to be a major subfamily, with both classical, and novel functions. In melanoma cell lines, for example, enhanced expression of the αVβ3 integrin correlated with tumorigenicity and metastatic properties (e.g. Felding-Habermann et al. 1992, J. Clin. Invest. 89, 2018; Marshall et al. 1991, Int. J. Cancer 49, 924), suggesting the involvement of αV-containing integrins in some steps of the tumor cell metastasis process. A similar role is discussed for carcinoma adhesion and spreading and colorectal cancer, respectively, which is one of the most common epithelial malignancies.
The αV-series integrins seem to exclusively recognize ligands bearing the RGD-(NH2-arginine-glycine-aspartic acid-COOH) tripeptide sequences, including those in vitronectin (αVβ1, αVβ3, αVβ5), fibronectin (αVβ1, αVβ3, αVβ5, αVβ6), and von Willebrand factor, fibrinogen, and osteopontin (αVβ3) (e.g., Busk et al. 1992, J. Biol. Chem. 267, 5790; Smith and Cheresh 1990, J. Biol. Chem. 265, 2168). Function blocking anti-integrin antibodies, e.g., antibodies directed against the αVβ3 integrin are also known (e.g., Cheresh and Spiro 1987, J. Biol. Chem. 262, 17703; Chuntharapai et al. 1993, Exp. Cell Res. 205, 345; EP 95 119 233).
The disruption of ligand-integrin interaction by peptides and antibodies has highlighted the important roles of αVβ3 integrin, “the vitronectin receptor,” processes as diverse as tumor growth and metastasis, viral infection, osteoporosis and angiogenesis (Felding-Habermann and Cheresh 1993, Curr. Opin. Cell Biol. 5, 864; Brooks et al. 1994, Cell 79, 1157; Brooks et al. 1994, Science 264, 569). The emergence of αVβ3 as a potential target for therapeutic intervention has thus led to a search for αVβ3 antagonists—a process that requires purified αVβ3. The source of biochemical amounts of αVβ3 has usually been human placental tissue. In common with most integral membrane proteins, integrins can only be obtained as active solubilized molecules in the presence of non-ionic detergents and therefore purification of αVβ3 from placenta for drug screening is both cumbersome, costly, and a considerable health risk (Mitjans et al. 1995, J. Cell Sci. 108, 2825).
Although recombinant integrins have been expressed already in eukaryotic systems, such as in CHO cells (O'Toole et al. 1990, Cell Regul. 1, 883) and in embryonic kidney 293 cells (King et al. 1994, J. Bone Miner Res. 9, 381) the preparation of biochemically satisfactory amounts has not been achieved by recombinant technology.
There have been no reports on the biological activity of truncated soluble αVβ3 constructs, and full-length αVβ3 receptor apparently has not been produced by recombinant technology in biochemically important amounts.
Thus, it would be desirable to provide a soluble recombinant αVβ3 receptor which can be produced in biochemically useful amounts and in a purified quality by a comparably easy and riskless process. Moreover, such a process would be also advantageous for producing full-length αVβ3 receptor.